ABCMdb

ABCB1 p.Leu976Cys

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Publications

PMID: 19456124 [PubMed] Crowley E et al: "Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1."

No. Sentence Comment

6 Two of the mutations (L976C and F978C) were found to reduce the binding of [γ-32 P]-azido-ATP to ABCB1. Login to comment
67 This necessitated the centrifugation (100000g for 30 min) of 500 μL Table 1: Mutagenic Oligonucleotide Primers Used To Generate TM12 Mutationsa mutation primer sequence (50 -30 ) diagnostic restriction digest L976C GAGGATGTTCTAtgtGTATTTTCAGCTGTTG -SpeI F978C GTTCTACTAGTATgTTCtGCaGTTGTCTTTGGTG +PstI A980C CTACTAGTATTTTCAtgcGTTGTCTTTGGTGCCATGGCC -PvuII V982C CTAGTATTTTCAGCgGTTtgCTTTGGTGCCATGGCC -PvuII G984C GCTGTTGTCTTTtGTGCtATGGCCGTGG -NcoI M986C GTATTTGGTGCttgtGCtGTGGGGCAAGTC -NcoI V988C GGTGCCATGGCCtgtGGGCAAGTCAGTTC -BstXI G989C CTTTGGTGCCATGGCCGTGtGcCAAGTCAGTTCATTTGC +BstXI Q990C GGCCGTGGGGtgtGTCtcTTCATTTGCTCC +EarI a Primer sequences contain an introduced cysteine residue (bold) and additional silent mutations (lower case), with respect to the coding sequence that generates or removes the indicated restriction site. Login to comment
118 The most striking alterations were the approximately 5-fold reductions in basal ATPase activity for the L976C (Vmax=26 ( 4 nmol min-1 mg-1 )and F978C (Vmax=27 ( 9 nmol min-1 mg-1 ) mutations compared to the control cysteine-less isoform of ABCB1. Login to comment
141 L976C (Vmax = 231 ( 80 nmol min-1 mg-1 ), F978C (Vmax = 142 ( 40 nmol min-1 mg-1 ), V988C, G989C, and Q990C all caused statisticallysignificant (p<0.05) reductionsinthe Vmax valuesfor nicardipine-stimulated ATPase activities (Figure 4B). Login to comment
149 For example, mutations L976C, F978C, V988C, and Q990C all displayed statistically significant reductions in maximal ATPase activity in the presence of vinblastine compared to the control cysteine-less isoform. Login to comment
155 Table 2: Potency and Degree of Drug Stimulation of ATP Hydrolysis by ABCB1a nicardipine vinblastine EC50 (μM) fold stimulation EC50 (μM) fold stimulation Cys-less 4.1 ( 1.1 4.0 ( 0.6 5.91 ( 2.9 2.2 ( 0.2 L976C 5.2 ( 0.2 7.4 ( 1.4 10.0 ( 0.0 3.5 ( 0.6 F978C 24.1 ( 2.3b 9.5 ( 1.4 42.9 ( 4.3b 2.3 ( 0.5 A980C 3.4 ( 0.3 5.1 ( 0.9 12.3 ( 1.8 3.2 ( 0.8 V982C 5.8 ( 0.9 4.2 ( 0.5 2.0 ( 0.7 1.8 ( 0.2 G984C 37.6 ( 11.2b 16.2 ( 6.6b 6.7 ( 1.7 6.2 ( 2.3 M986C 9.2 ( 0.8 4.7 ( 1.1 15.0 ( 2.0b 2.8 ( 0.7 V988C 3.9 ( 0.6 3.1 ( 0.1 7.3 ( 2.3 1.9 ( 0.2 G989C 13.6 ( 1.5 5.1 ( 1.6 4.9 ( 0.9 2.4 ( 0.3 Q990C 6.9 ( 1.1 3.7 ( 1.0 NDc NDc S992C 4.9 ( 0.5 4.2 ( 0.6 7.1 ( 2.6 2.3 ( 0.4 F994C 1.7 ( 0.4 3.2 ( 0.8 5.9 ( 2.5 1.6 ( 0.3 a ATPase activity was plotted as a function of the drug concentration and potency (EC50) and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation. Login to comment
172 Figure 5A presents a representative autoradiogram of [γ-32 P]-azido-ATP binding to the mutants L976C, F978C, A980C, V988C, G989C, and Q990C. Login to comment
174 The results indicate that at a concentration of 10 μM [γ-32 P]-azido-ATP there was a discernible difference between the binding of the ATP analogue to L976C, F978C, and A980C. Login to comment
175 Mutant isoforms L976C and F978C displayed a 4and 15-fold decrease in binding, whereas A980C showed a 1.8-fold increase in binding at the concentration of [γ-32 P]-azido-ATP used. Login to comment
178 Figure 5B shows the autoradiography data obtained for [γ-32 P]-azido-ATP photolabeling of the cysteine-less and L976C isoforms of ABCB1. Login to comment
179 Figure 5C shows the densitometric analysis of the dose-response curve for the cysteine-less, A980C, L976C, and F978C isoforms. Login to comment
181 In contrast, the mutant isoforms L976C (Bmax = 0.24, KD = 17 μM) and F978C (Bmax = 0.19, KD =17 μM) displayed 4-5-fold reductions in the amount of [γ-32 P]-azido-ATP binding compared to the cysteine-less control. Login to comment
182 Therefore, the decrease in basal and drug-stimulated ATPase activity observed for the L976C and F978C isoforms can be explained at least in part by impaired ATP binding at the NBDs. Login to comment
192 (B) Autoradiograms showing the binding of [γ-32 P]-azido-ATP (10-100 μM) to the purified, reconstituted cysteine-less and L976C mutant isoforms. Login to comment
193 (C) Data obtained for the cysteine-less, L976C, F978C, and A980C isoforms were analyzed by densitometry, and the amount bound was plotted as a function of the [γ-32 P]-azido-ATP concentration. Login to comment
220 The most dramatic effects in TM12 were observed in residues at the extracellular Table 3: Nucleotide Binding to ABCB1a ABCB1 isoform [32 P]-N3-ATP [32 P]-N3-ATP+ 1 mM ATP [32 P]-N3-ATP+ 1 mM ADP Cys-less 1.00 0.21 ( 0.05 0.23 ( 0.06 L976C 0.21 ( 0.05 0.10 ( 0.02 0.05 ( 0.03 F978C 0.07 ( 0.01 ND ND A980C 1.81 ( 0.71 0.45 ( 0.10 0.15 ( 0.08 V988C 0.53 ( 0.20 ND ND G989C 0.83 ( 0.04 0.10 ( 0.05 0.13 ( 0.06 Q990C 1.05 ( 0.30 0.19 ( 0.11 0.01 ( 0.01 a The ABCB1 isoforms were incubated with 10 μM [γ32 P]-azido-ATP in the presence or absence of excess unlabeled nucleotides (1 mM). Login to comment
232 (L976C and F978C) and intracellular (V988C and Q990C) ends of the helix. Login to comment
234 In contrast, the reduced basal activity observed with mutants L976C and F978C appeared to be a consequence of reduced nucleotide binding. Login to comment

PMID: 20731718 [PubMed] Crowley E et al: "Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1."

No. Sentence Comment

63 The lowest labelling observed in the selection of TM12 mutant isoforms was at L976C, with an Lext of 38 ± 5%. Login to comment
82 The rate of labelling (i.e. t1 / 2) was divided into fast (L986C- G992C, average t1 / 2 $ 8 min) and slow (L976C- G984C, average t1 / 2 $ 25 min) kinetics between the carboxy-half and the amino-half, respectively. Login to comment
118 Conformational changes - amino region of TM12 As shown in Table 2, the amino region of TM12 (L976C-V982C) was not associated with large alterations in topography. Login to comment
121 For example, L976C became less accessible to BM, but more accessible to CM, following a shift from the basal to the nucleotide-bound conformation. Login to comment
139 Mutant CM BM FM Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) L976C 38 ± 5 29 ± 12 66 ± 14 29 ± 18 - - A980C 53 ± 6 34 ± 1 54 ± 8 20 ± 9 - - V982C 98 ± 14 15 ± 6 164 ± 50 27 ± 17 - - G984C 73 ± 14 29 ± 6 84 ± 24 22 ± 7 13 ± 10 ND M986C 89 ± 30 25 ± 10 51 ± 5 3 ± 2 21 ± 2 ND V988C 53 ± 6 37 ± 18 221 ± 63 18 ± 12 - - G989C 64 ± 7 15 ± 6 21 ± 3 9 ± 2 - - S992C 55 ± 4 22 ± 6 51 ± 5 4 ± 1 32 ± 3 25 ± 5 F994C 51 ± 10 11 ± 9 111 ± 35 13 ± 10 129 ± 24 8 ± 3 Conformational changes - central region Two of the residues examined in the central region (G984C and M986C) of TM12 have been shown to accommodate partial labelling with FM, suggestive of aqueous accessibility in the basal state. At M986C, the extent of labelling with the hydrophilic probe was increased following the addition of nonhydrolysable nucleotide. Login to comment
164 ABCB1 isoform Catalytic intermediate CM BM FM L976C Basal ++ +++ ) AMP-PNP +++ ++ ) Vi trapped +++ +++ ) A980C Basal ++ ++ ) AMP-PNP +++ + ) Vi trapped +++ +++ ) V982C Basal +++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G984C Basal +++ +++ + AMP-PNP +++ +++ + Vi trapped +++ ++ ) M986C Basal +++ ++ + AMP-PNP ++ +++ ++ Vi trapped +++ ++ ) V988C Basal ++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G989C Basal ++ + ) AMP-PNP ++ ++ ) Vi trapped ++ + ) S992C Basal ++ ++ + AMP-PNP +++ +++ ++ Vi trapped ++ ++ + F994C Basal ++ +++ +++ AMP-PNP ++ +++ ++ Vi trapped +++ +++ + reflect localization at the membrane-solute interface. Login to comment

PMID: 9261097 [PubMed] Loo TW et al: "Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12."

No. Sentence Comment

80 We also tested mutants F335C/L976C, L339C/S979C, F343C/F983C, G347C/A987C, and S351C/ V991C for cross-linking since they were predicted to lie on opposing faces of TM6 and TM12 modeled in a right-handed coiled-coil. Login to comment